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In vivo kinetics of protein targeting to the endoplasmic reticulum determined by site‐specific phosphorylation
Author(s) -
Goder Veit,
Crottet Pascal,
Spiess Martin
Publication year - 2000
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/19.24.6704
Subject(s) - endoplasmic reticulum , biology , cytosol , phosphorylation , microbiology and biotechnology , n terminus , translation (biology) , biochemistry , peptide sequence , messenger rna , enzyme , gene
We have developed a novel assay to detect the cytosolic localization of protein domains by inserting a short consensus sequence for phosphorylation by protein kinase A. In transfected COS‐1 cells, this sequence was labeled efficiently with [ 32 P]phosphate only when exposed to the cytosol and not when translocated into the lumen of the endoplasmic reticulum. The phosphorylation state of this sequence can therefore be used to determine the topology of membrane proteins. This assay is sufficiently sensitive to detect even the transient cytosolic exposure of the N‐terminal domain of a membrane protein with a reverse signal‐anchor sequence. The extent of phosphorylation per newly synthesized polypeptide was shown to reflect the time of exposure to the cytosol, which depends on translation, targeting and translocation of the N‐terminus. By altering the length of the N‐terminal domain or manipulating the translation rate, it was determined that protein targeting is rapid and requires only a few seconds. The rate of N‐terminal translocation was estimated to be ∼1.6 times the rate of translation.